This is the competing renewal of a research program that examines the biophysical and molecular basis of cholinergic inhibition of cochlear hair cells. The neurotransmitter acetylcholine (ACh) opens ionotropic (cation-permeant) receptors (AChRs) composed of 19 and 110 subunits. Calcium influx through the AChR activates calcium-sensitive potassium channels (encoded by the SK2 gene) to hyperpolarize and inhibit the hair cell. While calcium influx through the AChR seems adequate to activate the SK channels, other observations suggest that calcium may be released from endoplasmic stores to extend and amplify that process. The structure of the efferent synapse includes a near-membrane cistern in the hair cell that may serve as a calcium store. Genetic alteration of the hair cell's AChR and associated SK channels has provided some insight into efferent function. In particular, alteration of a single amino acid in the permeation path of 19 produced a 'super receptor' with prolonged open times, greater sensitivity to ACh and slowed desensitization. In the knockin mouse possessing this receptor, efferent inhibition was greatly enhanced, and the animal was better protected from permanent loud sound damage. We will continue to probe the molecular mechanisms of cholinergic inhibition in this and other transgenic models. In addition, we will exploit functional and genetic differences between avian (chicken) and mammalian AChRs to explore the role of calcium flux in detail. Should progress warrant it, we will construct a transgenic mouse substituted with chicken 19. We will perform detailed ultrastructural analyses of synaptic cisterns in wildtype and transgenic mice lacking one or more components of the efferent synapse. The small cytoplasmic gap between cistern and postsynaptic membrane is replete with organized electron-densities, suggesting the presence of molecular 'connectors' that may include the cytoplasmic portions of AChRs and calcium release channels. This arrangement is strongly reminiscent of junctional complexes in muscle and suggests the possibility of 'conformational coupling' between AChRs and calcium release channels, in addition to calcium-based signaling. Efferent innervation of the inner ear modulates sensitivity and protects against acoustic trauma. Our growing knowledge of the molecular bases for this process has begun to reveal potential therapeutic targets, supported by the unique pharmacology of hair cell acetylcholine receptors. Thorough knowledge of the molecular bases of cholinergic inhibition will advance therapeutic strategies exploiting those mechanisms.